Primase plays a key role in DNA replication by synthesizing a short RNA oligonucleotide "primer onto which polymerase alpha can polymerize multiple deoxynucleotides. Primase is composed of a p49 subunit that contains catalytic activity and a p58 subunit whose function is less clear. To better understand the role of p58, the overall objective of this work is to identify regions in human p58 important to primase activity. Of particular interest is a sequence in p58 which has high homology with the 8 kDa DNA binding domain of polymerase beta. My hypothesis is that this highly conserved "pol beta-like" motif is important to primer synthesis, possibly through interaction with template or NTPs. To address this hypothesis, a series of mutant p58 proteins will be constructed using site-directed mutagenesis and PCR techniques; (1) p58 mutant that lacks the "pol beta-like" motif, (2) "pol beta-like" motif expressed alone, and (3) p58 mutants containing single-site mutations. For each mutant, the binding to p48 subunit, NTPs and template will be assessed. The relationship between these properties and enzymatic activity will provide a more complete picture of p58 contribution to primase mechanism. Additionally, identification of common characteristics between primase p58 and the 8 kDa domain of polymerase beta would provide evidence for a close evolutionary link between these two proteins. Ultimately, this knowledge can provide the basis for developing pharmacological strategies to target primase initiation of new DNA strands, an attractive strategy for selectively killing rapidly dividing cancer cells.